The use of specific gene targeting in eukaryotic cell-based model systems provides an effective and selective strategy for studying the function of a particular gene in response to biological or chemical molecules as well as for model systems to produce biochemicals for therapeutic use. In particular is the use of homologous recombination to: (1) inactivate gene function to study downstream functions; (2) introduce reporter gene molecules into targeted loci to facilitate the screening of gene expression in response to biomolecules and/or pharmaceutical compounds; (3) generate stable, steady-state expression of target genes via the introduction of constitutively active heterologous promoter elements or through chromosomal site-specific gene amplification.
Standard methods for introducing targeting genes to a locus of interest are known by those skilled in the art. Gene targeting in prokaryotes and lower organisms has been well established, and methods for in vivo gene targeting in animal models have also been described (de Wind N. et al. (1995) “Inactivation of the mouse Msh2 gene results in mismatch repair deficiency, methylation tolerance, hyperrecombination, and predisposition to cancer” Cell 82:321-300).
The generation of knockouts in somatic cells, however, is more problematic due to low efficiency of transfection and endogenous biochemical activities that monitor for DNA strand exchange. Work done by Waldman et al. (Waldman, T., Kinzler, K. W., and Vogelstein, B. (1995) Cancer Res. 55:5187-5190) demonstrated the ability to generate somatic cell knockouts in a human cell line called HCT116 at relatively high rate. In the described studies, the authors used a targeting vector containing the neomycin (neo) resistance gene to knockout a locus of interest. Using this cell line the authors reported 37% of the neo resistant clones tested were found to contain a targeting vector within the homologous locus in the genome of the host.
Similar studies using other cell lines by these authors have been less successful. While the reason(s) for the lack or significant reduction in the frequency of recombination in somatic cell lines are not clear, some factors, such as the degree of transfection as well as the differences that may occur within the intracellular milieu of the host may play critical roles with regard to recombination efficiency. In the studies performed by Waldman et al., the cell line that the authors used was inherently defective for mismatch repair (MMR), a process involved in monitoring homologous recombination (de Wind N. et al. (1995) Cell 82:321-300). One proposed method for the high degree of recombination in this line was the lack of MMR, which has been implicated as a critical biochemical pathway for monitoring recombination (Reile, T E et al. WO 97/05268; Rayssigguier, C., et al. (1989) Nature 342:396-401; Selva, E., et al. (1995) Genetics 139:1175-1188; U.S. Pat. No. 5,965,415 to Radman). Indeed, studies using mammalian and prokaryotic cells defective for MMR have previously demonstrated the increased chromosomal recombination with DNA fragments having up to 30% difference in sequence identity.
Nevertheless, homologous recombination in mammalian somatic cell lines has been and remains problematic due to the low efficiency of recombination. Although it is believed by many skilled in the art that low rate of homologous recombination may be overcome by the blockade of MMR (Reile, T E et al. WO 97/05268; Rayssigguier, C., et al. (1989) Nature 342:396-401; Selva, E., et al. (1995) Genetics 139:1175-1188; U.S. Pat. No. 5,965,415 to Radman; Beth Elliott and Maria Jasin, “Repair of Double-Strand Breaks by Homologous Recombination in Mismatch Repair-Defective Mammalian Cells” (2001) Mol. Cell Biol., 21:2671-2682) these methods teach the use of using MMR defective unicellular organisms to increase homologous recombination. A significant bottleneck to this approach is the need to clone large segments of homologous DNA from the target locus. Moreover, while it has been reported that short oligonucleotides are capable of homologously recombining at site-specific regions of the genome (Igoucheva O, Alexeev V, Yoon K., (2001) “Targeted gene correction by small single-stranded oligonucleotides in mammalian cells” Gene Ther. 8:391-399), the ability to integrate larger fragments with short terminal regions of homology remains elusive. In fact, recent studies by Inbar et al. (Inbar O, Liefshitz B, Bitan G, Kupiec M., (2000) “The Relationship between Homology Length and Crossing Over during the Repair of a Broken Chromosome” J. Biol. Chem. 275:30833-30838) demonstrated that fragments that contained only 123 bps of homologous sequence were not sufficient to induce homologous exchange of large DNA fragments in yeast. It has not been heretofore demonstrated that larger DNA fragments, such as those containing regulated or constitutively active promoter elements, gene inserts or reporter genes could be integrated into the exon of a locus in somatic mammalian cell lines with short, homologous terminal ends, such as fragments of only 20-120 nucleotides.